Replication of the eukaryotic chromosome is an ordered process involving the activation of sets of replication origins at different times during the S phase. The activation of multiple replication origins on each chromosome insures that every chromosome replicates during each cell cycle. An especially rigorous regulatory mechanism prevents second activation of origins thereby preventing chromosome imbalance. Understanding how cells impose these controls will require much more information about the rules that govern origin activation in vivo. Our work with the yeast Saccharomyces cerevisae has begun to reveal some of these rules. We propose to examine four areas of control of replication origin activation. (1) Placing origins in close proximity results in reduction in their efficiency, and dispensible, non-origin sequences influence which origin is activated. We will test models for interference and determine the mechanism that biases origin selection. (2) The proximity to a telomere causes an origin's activation to be delayed until late S phase. We will determine whether maintenance of the "late-context" requires continuous physical attachment of the telomere to the chromosome and when in the cell cycle the context is established. (3) Early origin activation can be suppressed by a non-telomeric, cis- acting element that is independent of the telomere. We will characterize this element and use it to determine the effect of altered timing on chromosome stability. We will define the size of late replicating domains. (4) Eukaryotic chromosomal origins appear to be confined exclusively to intergenic DNA. Using S. cerevisiae and Arabidopsis thaliana we will test the hypothesis that initiations are confined to intergenic sequences by properties of gene boundaries. This work will lead to a greater understanding of the regulation of chromosome replication in normal cells and in those with defective growth properties, such as cancer cells.